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PECAM-1 (CD31) homophilic interaction up-regulates alpha6beta1 on transmigrated neutrophils in vivo and plays a functional role in the ability of alpha6 integrins to mediate leukocyte migration through the perivascular basement membrane.

Dangerfield J, Larbi KY, Huang MT, Dewar A, Nourshargh S - J. Exp. Med. (2002)

Bottom Line: Platelet-endothelial cell adhesion molecule (PECAM)-1 has been implicated in leukocyte migration through the perivascular basement membrane (PBM) though the mechanisms involved are unclear.An anti-alpha(6) integrins mAb (GoH3) inhibited (78%, P < 0.001) neutrophil migration through interleukin (IL)-1beta-stimulated cremasteric venules, primarily at the level of the PBM, as analyzed by intravital and electron microscopy.In PECAM-1-deficient mice (KO), a reduced level of neutrophil transmigration elicited by IL-1beta (4-h reaction) was observed in both the cremaster muscle (55% inhibition, P < 0.05) and in the peritoneum (57% inhibition, P < 0.01) but GoH3 had no additional inhibitory effect on these responses.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Medicine Unit, National Heart & Lung Institute, Faculty of Medicine, Imperial College, Hammersmith Hospital, London W12 ONN, United Kingdom.

ABSTRACT
Platelet-endothelial cell adhesion molecule (PECAM)-1 has been implicated in leukocyte migration through the perivascular basement membrane (PBM) though the mechanisms involved are unclear. The present results demonstrate that the ability of alpha(6) integrins to mediate neutrophil migration through the PBM is PECAM-1 dependent, a response associated with PECAM-1-mediated increased expression of alpha(6)beta(1) on transmigrating neutrophils in vivo. An anti-alpha(6) integrins mAb (GoH3) inhibited (78%, P < 0.001) neutrophil migration through interleukin (IL)-1beta-stimulated cremasteric venules, primarily at the level of the PBM, as analyzed by intravital and electron microscopy. In PECAM-1-deficient mice (KO), a reduced level of neutrophil transmigration elicited by IL-1beta (4-h reaction) was observed in both the cremaster muscle (55% inhibition, P < 0.05) and in the peritoneum (57% inhibition, P < 0.01) but GoH3 had no additional inhibitory effect on these responses. FACS((R)) analysis of neutrophils demonstrated increased expression of alpha(6)beta(1) on transmigrated peritoneal neutrophils, as compared with blood neutrophils, in wild-type but not KO mice even though neutrophils from both strains of mice exhibited comparable levels of intracellular expression of alpha(6) as observed by immunofluorescent staining and confocal microscopy. Furthermore, mice deficient in either leukocyte or endothelial cell PECAM-1, as developed by bone marrow transplantation, demonstrated a similar level of reduced neutrophil transmigration and expression of alpha(6)beta(1) on transmigrated neutrophils as that detected in KO mice. The results demonstrate a role for PECAM-1 homophilic interaction in neutrophil transmigration and increased expression of alpha(6)beta(1) on the cell surface of transmigrated neutrophils in vivo, a response that could contribute to the mechanism of PECAM-1-mediated neutrophil migration through the PBM.

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Cell surface expression of α6β1, PECAM-1, and L-selectin on blood and IL-1β–elicited peritoneal neutrophils in WT and PECAM-1 deficient (KO) mice. Panel A shows representative fluorescence histograms comparing cell surface expressions of α6 and β1 on blood and IL-1β–induced peritoneal neutrophils in WT and PECAM-1 deficient mice, as indicated. The filled tracings are from blood samples incubated with an isotype-matched control mAb (the same low binding was found with peritoneal samples) and the solid and dashed line tracings are from blood and peritoneal samples, respectively, incubated with mAbs GoH3 (anti-α6) or 9EG7 (anti-β1), as indicated. B shows pooled relative fluorescence intensities of samples stained with specific antibodies and analyzed and quantified by FACS® as detailed in Materials and Methods. The data represent mean ± SEM of samples obtained from n = 3–10 animals/group. Significant binding of primary mAbs is indicated by asterisks, *P < 0.05, **P < 0.01 and other statistical comparisons are indicated by lines.
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fig4: Cell surface expression of α6β1, PECAM-1, and L-selectin on blood and IL-1β–elicited peritoneal neutrophils in WT and PECAM-1 deficient (KO) mice. Panel A shows representative fluorescence histograms comparing cell surface expressions of α6 and β1 on blood and IL-1β–induced peritoneal neutrophils in WT and PECAM-1 deficient mice, as indicated. The filled tracings are from blood samples incubated with an isotype-matched control mAb (the same low binding was found with peritoneal samples) and the solid and dashed line tracings are from blood and peritoneal samples, respectively, incubated with mAbs GoH3 (anti-α6) or 9EG7 (anti-β1), as indicated. B shows pooled relative fluorescence intensities of samples stained with specific antibodies and analyzed and quantified by FACS® as detailed in Materials and Methods. The data represent mean ± SEM of samples obtained from n = 3–10 animals/group. Significant binding of primary mAbs is indicated by asterisks, *P < 0.05, **P < 0.01 and other statistical comparisons are indicated by lines.

Mentions: As GoH3 inhibited neutrophil migration in WT but not PECAM-1–deficient mice, we investigated the expression of α6β1 on the cell surface of neutrophils in the two strains of animals. With respect to blood neutrophils, low levels of cell surface α6 and β1 were detected in WT and PECAM-1–deficient mice (Fig. 4, A and B). Similarly, low levels of PECAM-1 (Fig. 4 B) and α4 integrins (unpublished data) were detected, although high levels of L-selectin (Fig. 4 B) and β2 integrins were observed (unpublished data). Overall, no marked differences in expressions of adhesion molecules were detected between WT and PECAM-1–deficient blood neutrophils. With respect to IL-1β–induced peritoneal neutrophils, a significantly enhanced level of α6 and β1 expression was detected on cells obtained from WT but not PECAM-1–deficient mice (Fig. 4, A and B), indicating an association between cell surface expression of α6β1 on transmigrated neutrophils and PECAM-1, in vivo. By comparison, cell surface expression of L-selectin was equally reduced on transmigrated neutrophils, as compared with blood neutrophils, in both WT and KO mice (Fig. 4 B).


PECAM-1 (CD31) homophilic interaction up-regulates alpha6beta1 on transmigrated neutrophils in vivo and plays a functional role in the ability of alpha6 integrins to mediate leukocyte migration through the perivascular basement membrane.

Dangerfield J, Larbi KY, Huang MT, Dewar A, Nourshargh S - J. Exp. Med. (2002)

Cell surface expression of α6β1, PECAM-1, and L-selectin on blood and IL-1β–elicited peritoneal neutrophils in WT and PECAM-1 deficient (KO) mice. Panel A shows representative fluorescence histograms comparing cell surface expressions of α6 and β1 on blood and IL-1β–induced peritoneal neutrophils in WT and PECAM-1 deficient mice, as indicated. The filled tracings are from blood samples incubated with an isotype-matched control mAb (the same low binding was found with peritoneal samples) and the solid and dashed line tracings are from blood and peritoneal samples, respectively, incubated with mAbs GoH3 (anti-α6) or 9EG7 (anti-β1), as indicated. B shows pooled relative fluorescence intensities of samples stained with specific antibodies and analyzed and quantified by FACS® as detailed in Materials and Methods. The data represent mean ± SEM of samples obtained from n = 3–10 animals/group. Significant binding of primary mAbs is indicated by asterisks, *P < 0.05, **P < 0.01 and other statistical comparisons are indicated by lines.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2194111&req=5

fig4: Cell surface expression of α6β1, PECAM-1, and L-selectin on blood and IL-1β–elicited peritoneal neutrophils in WT and PECAM-1 deficient (KO) mice. Panel A shows representative fluorescence histograms comparing cell surface expressions of α6 and β1 on blood and IL-1β–induced peritoneal neutrophils in WT and PECAM-1 deficient mice, as indicated. The filled tracings are from blood samples incubated with an isotype-matched control mAb (the same low binding was found with peritoneal samples) and the solid and dashed line tracings are from blood and peritoneal samples, respectively, incubated with mAbs GoH3 (anti-α6) or 9EG7 (anti-β1), as indicated. B shows pooled relative fluorescence intensities of samples stained with specific antibodies and analyzed and quantified by FACS® as detailed in Materials and Methods. The data represent mean ± SEM of samples obtained from n = 3–10 animals/group. Significant binding of primary mAbs is indicated by asterisks, *P < 0.05, **P < 0.01 and other statistical comparisons are indicated by lines.
Mentions: As GoH3 inhibited neutrophil migration in WT but not PECAM-1–deficient mice, we investigated the expression of α6β1 on the cell surface of neutrophils in the two strains of animals. With respect to blood neutrophils, low levels of cell surface α6 and β1 were detected in WT and PECAM-1–deficient mice (Fig. 4, A and B). Similarly, low levels of PECAM-1 (Fig. 4 B) and α4 integrins (unpublished data) were detected, although high levels of L-selectin (Fig. 4 B) and β2 integrins were observed (unpublished data). Overall, no marked differences in expressions of adhesion molecules were detected between WT and PECAM-1–deficient blood neutrophils. With respect to IL-1β–induced peritoneal neutrophils, a significantly enhanced level of α6 and β1 expression was detected on cells obtained from WT but not PECAM-1–deficient mice (Fig. 4, A and B), indicating an association between cell surface expression of α6β1 on transmigrated neutrophils and PECAM-1, in vivo. By comparison, cell surface expression of L-selectin was equally reduced on transmigrated neutrophils, as compared with blood neutrophils, in both WT and KO mice (Fig. 4 B).

Bottom Line: Platelet-endothelial cell adhesion molecule (PECAM)-1 has been implicated in leukocyte migration through the perivascular basement membrane (PBM) though the mechanisms involved are unclear.An anti-alpha(6) integrins mAb (GoH3) inhibited (78%, P < 0.001) neutrophil migration through interleukin (IL)-1beta-stimulated cremasteric venules, primarily at the level of the PBM, as analyzed by intravital and electron microscopy.In PECAM-1-deficient mice (KO), a reduced level of neutrophil transmigration elicited by IL-1beta (4-h reaction) was observed in both the cremaster muscle (55% inhibition, P < 0.05) and in the peritoneum (57% inhibition, P < 0.01) but GoH3 had no additional inhibitory effect on these responses.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Medicine Unit, National Heart & Lung Institute, Faculty of Medicine, Imperial College, Hammersmith Hospital, London W12 ONN, United Kingdom.

ABSTRACT
Platelet-endothelial cell adhesion molecule (PECAM)-1 has been implicated in leukocyte migration through the perivascular basement membrane (PBM) though the mechanisms involved are unclear. The present results demonstrate that the ability of alpha(6) integrins to mediate neutrophil migration through the PBM is PECAM-1 dependent, a response associated with PECAM-1-mediated increased expression of alpha(6)beta(1) on transmigrating neutrophils in vivo. An anti-alpha(6) integrins mAb (GoH3) inhibited (78%, P < 0.001) neutrophil migration through interleukin (IL)-1beta-stimulated cremasteric venules, primarily at the level of the PBM, as analyzed by intravital and electron microscopy. In PECAM-1-deficient mice (KO), a reduced level of neutrophil transmigration elicited by IL-1beta (4-h reaction) was observed in both the cremaster muscle (55% inhibition, P < 0.05) and in the peritoneum (57% inhibition, P < 0.01) but GoH3 had no additional inhibitory effect on these responses. FACS((R)) analysis of neutrophils demonstrated increased expression of alpha(6)beta(1) on transmigrated peritoneal neutrophils, as compared with blood neutrophils, in wild-type but not KO mice even though neutrophils from both strains of mice exhibited comparable levels of intracellular expression of alpha(6) as observed by immunofluorescent staining and confocal microscopy. Furthermore, mice deficient in either leukocyte or endothelial cell PECAM-1, as developed by bone marrow transplantation, demonstrated a similar level of reduced neutrophil transmigration and expression of alpha(6)beta(1) on transmigrated neutrophils as that detected in KO mice. The results demonstrate a role for PECAM-1 homophilic interaction in neutrophil transmigration and increased expression of alpha(6)beta(1) on the cell surface of transmigrated neutrophils in vivo, a response that could contribute to the mechanism of PECAM-1-mediated neutrophil migration through the PBM.

Show MeSH
Related in: MedlinePlus